The AAA family of proteins is involved in a wide variety of cellular processes. One of the best-characterized members of the AAA family is NSF which has an ATP-dependent role in vesicle fusion. An NSF substrate trap has been created by generating an ATP-hydrolysis-deficient mutant. The substrate range of NSF has been at least partially defined. The goal of this proposal is to employ NSF substrate traps in cells to define the range/specificity of substrate recognition by NSF. In order to identify NSF's substrate binding site, a structure-based mutagenesis analysis of the N-domain of NSF will be performed and the substrate trap's ability to bind targets will be tested. The development of the substrate trap method will lay the groundwork for extending our analysis of NSF substrate binding to other AAA proteins such as "orphan" AAA proteins, for which the substrate range is completely unknown. Some orphan AAA proteins are associated with diseases. The specific aims of this proposal are to use NSF substrate traps (1) to develop the AAA trap methodology with NSF, a protein for which at least some substrates have been identified. (2) to determine whether NSF has a broader substrate range than previously appreciated. (3) to carry out mutagenic analysis to determine the structural elements within NSF's N-terminal domain that are critical for substrate recognition.